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Summary

The C. elegans PUF and FBF proteins regulate various aspects of
germline development by selectively binding to the 3′ untranslated
region of their target mRNAs and repressing translation. Here, we show that
puf-8, fbf-1 and fbf-2 also act in the soma where they
negatively regulate vulvaI development. Loss-of-function mutations in
puf-8 cause ectopic vulval differentiation when combined with
mutations in negative regulators of the EGFR/RAS/MAPK pathway and suppress the
vulvaless phenotype caused by mutations that reduce EGFR/RAS/MAPK signalling.
PUF-8 acts cell-autonomously in the vulval cells to limit their temporal
competence to respond to the extrinsic patterning signals. fbf-1 and
fbf-2, however, redundantly inhibit primary vulval cell fate
specification in two distinct pathways acting in the soma and in the germline.
The FBFs thereby ensure that the inductive signal selects only one vulval
precursor cell for the primary cell fate. Thus, translational repressors
regulate various aspects of vulval cell fate specification, and they may play
a conserved role in modulating signal transduction during animal
development.

INTRODUCTION

The spatial and temporal regulation of gene expression can occur either at
the level of gene transcription or at the level of mRNA export, stability or
translation through RNA-binding proteins or micro RNAs
(de Moor et al., 2005;
Kuersten and Goodwin, 2003).
Work on model organisms such as Drosophila melanogaster and
Caenorhabditis elegans has contributed much to our current
understanding of post-transcriptional gene regulation during development.
Translational control by RNA binding proteins is frequently used in the C.
elegans germline and early embryo, but translational regulation has also
been observed during larval development
(Kuersten and Goodwin, 2003;
Rougvie, 2001). Many mRNAs
contain sequence motifs in their 5′ or 3′ untranslated regions
(5′UTRs or 3′UTRs) that serve as binding sites for regulatory
proteins controlling different aspects of mRNA localization, translation or
stability.

The PUF gene family is conserved from yeast to humans. PUF proteins
function as translational repressors that bind to specific elements in the
3′UTRs of their target mRNAs (reviewed by
Wickens et al., 2002). The
first characterized members of this family were Drosophila Pumilio
and the two C. elegans FBF proteins. Hence, this family is referred
to as PUF for Pumilio and FBF repeat proteins
(Zhang et al., 1997). Typical
PUF proteins contain eight PUF repeats of approximately 40 amino acids with a
core consensus sequence containing aromatic and basic residues. The PUF
repeats directly bind to the target mRNAs and recruit additional proteins such
as Nanos, Brain tumor and CPEB (Kraemer et
al., 1999; Luitjens et al.,
2000; Sonoda and Wharton,
1999; Sonoda and Wharton,
2001). The cis-regulatory elements in the 3′ UTRs of their
target mRNAs contain a UGUR tetra nucleotide sequence motif termed a
Nanos response element (NRE). The binding specificity of the
individual PUF proteins is thought to be determined by additional flanking
nucleotides (Murata and Wharton,
1995; Tadauchi et al.,
2001; Wharton et al.,
1998; Zamore et al.,
1997; Zhang et al.,
1997).

Pumilio, the only PUF protein in Drosophila melanogaster,
controls, together with Nanos, the establishment of the anterior-posterior
axis of the embryo by repressing the translation of maternal
hunchback mRNA (Barker et al.,
1992; Murata and Wharton,
1995). Pumilio and Nanos also inhibit cyclin B
translation in migrating pole cells allowing them to arrest in G2 until they
reach the gonads (Asaoka-Taguchi et al.,
1999). In addition to its roles during development,
Drosophila Pumilio was recently shown to be necessary for the
activity-dependent expression of the voltage-gated sodium channel Paralytic in
the central nervous system (Mee et al.,
2004). The human and mouse genomes each encode two PUF proteins
with unknown functions (Spassov and
Jurecic, 2002; Spassov and
Jurecic, 2003).

The C. elegans genome contains the surprisingly high number of
eleven PUF genes (fbf-1 and fbf-2, puf-3 to
puf-11). PUF-8 forms, together with PUF-9, a distinct subgroup among
the C. elegans PUF proteins, as PUF-8 and PUF-9 are more similar to
the Drosophila and to the two vertebrate pumilio proteins than to the
other C. elegans PUF proteins
(Wickens et al., 2002). FBF-1
and FBF-2 (fem-3-binding factor-1 and -2) are two closely related
proteins that regulate the sperm/oocyte switch in the hermaphrodite germline
by binding to the PME (point mutation element) in the 3′ UTR of
fem-3 mRNA (Ahringer and Kimble,
1991; Kraemer et al.,
1999; Zhang et al.,
1997). In addition, FBF-1 and FBF-2 both regulate the mitosis
versus meiosis decision in the distal region of the germline by repressing
gld-1 translation in the mitotic region to prevent the stem cells
from entering meiosis (Crittenden et al.,
2002; Kadyk and Kimble,
1998). Furthermore, FBF and PUF proteins are required for germ
cell survival, germ cell migration and the mitotic arrest of germ cells during
embryogenesis (Kraemer et al.,
1999; Subramaniam and Seydoux,
1999). PUF-8 is necessary for the meiotic division of the primary
spermatocytes in hermaphrodites and males
(Subramaniam and Seydoux,
2003).

Here, we show that the same PUF proteins that control germline development
also act in the soma during vulval induction. During larval development, the
hermaphrodite vulva is formed out of 22 cells that are generated by three out
of six equivalent vulval precursor cells (VPCs; P3.p through P8.p)
(Greenwald, 1997). To induce
vulval differentiation, the anchor cell (AC) in the somatic gonad sends an
epidermal growth factor signal (LIN-3) to the adjacent VPCs
(Hill and Sternberg, 1992).
This inductive AC signal activates the LET-23 EGFR signalling pathway in the
nearest VPC (P6.p) to specify the primary (1°) cell fate. P6.p then sends
a lateral signal to the neighbouring VPCs, P5.p and P7.p, via the LIN-12 NOTCH
pathway (Greenwald et al.,
1983; Sternberg,
1988). LIN-12 signalling inhibits the 1° fate specification in
P5.p and P7.p and instead instructs the secondary (2°) fate in these cells
(Ambros, 1999;
Sternberg, 1988). Multiple
inhibitory signalling pathways antagonize the EGFR/RAS/MAPK pathway to control
the cell fate choice in the VPCs (reviewed by
Fay and Han, 2000). These
inhibitors ensure that the distal VPCs (P3.p, P4.p and P8.p), which receive
little or no inductive and lateral signals, adopt the tertiary (3°)
non-vulval cell fate. After the vulval cell fates have been specified, the
VPCs undergo stereotypic patterns of cell divisions before they differentiate
and form the mature organ. Three rounds of symmetric cell divisions generate
eight 1° descendants, of which four adopt the VulE and four the VulF
subfate. The last of the three cell divisions in the 2° lineage generates
only seven descendants that further differentiate into the VulA, VulB, VulC
and VulD subfates (Inoue et al.,
2002; Sternberg and Horvitz,
1986). The 3° cells divide only once and then fuse with the
surrounding hypodermal syncytium (hyp7).

Our analysis indicates that puf-8, fbf-1 and fbf-2
negatively regulate vulval induction in parallel with the known inhibitors of
the EGFR/RAS/MAPK pathway. puf-8 restricts the temporal competence of
the vulval cells by promoting the fusion of the uninduced 3° cells with
hyp7, while fbf-1 and fbf-2 control the 1° versus
2°/3° cell fate decision.

MATERIALS AND METHODS

Nematode strains and general methods

All strains were derivatives of Bristol strain N2 of Caenorhabditis
elegans and grown under standard conditions at 20°C
(Brenner, 1974) or at the
temperature indicated in the table footnotes. Unless noted otherwise, the
mutations used have been described previously
(Riddle et al., 1997) and are
listed below by their linkage group.

GFP and YFP expression was observed under fluorescent light illumination
with a Leica DMRA microscope equipped with a cooled CCD camera (Hamamatsu
ORCA-ER) controlled by the Openlab 3.0 software (Improvision). Animals were
mounted on 3% agarose pads in M9 solution containing 15 mM NaN3.
Larvae were first inspected using Nomarski optics to identify the position of
the Pn.p cells or their descendants, and GFP or YFP expression was then scored
under fluorescent light illumination using the same exposure settings for a
particular transgene in all different genetic backgrounds. For the PUF-8::GFP
FBF-2::GFP and the EGL-17::YFP experiments, three semi-quantitative classes
were made: no expression if the fluorescence was not distinguishable from the
background staining, low expression if there was a weak but clearly visible
signal, and high expression if the fluorescence signal was strong. The images
of PUF-8::GFP and FBF-2::GFP at the L4 stages needed a correction to prevent
overexposure.

The induction index of the VPCs was scored under Nomarski optics and the
average number of 1° or 2° induced VPCs per animal was calculated as
described previously (Dutt et al.,
2004).

Laser ablation of the somatic gonad precursors Z1 and Z4 and germline
precursors Z2 and Z3 were done as described by Kimble
(Kimble, 1981), and induction
was scored in L4 larvae.

Non-complementation screen to isolate puf-8(zh17):
gap-1(ga133) males were mutagenized with EMS as described above,
mated with unc-4(e120) puf-8(ga145); gap-1(ga133) hermaphrodites and
the nonUnc F1 progeny was screened for Muv animals. After screening 2,000
haploid genomes one Muv non-Unc animal was identified and propagated.
ga145 was mapped with three-factor mapping between dpy-10
and unc-4 on LGII and further narrowed down by transformation rescue
experiments using YACs and cosmids to the cosmid clone C30G12. RNAi analysis
of the genes encoded by C30G12 in a gap-1(ga133) background
identified the puf-8 gene as candidate, and DNA sequencing of the
puf-8 coding region in the ga145 and zh17 alleles
identified the molecular lesions.

RNA interference analysis

RNA interference analysis (RNAi) was performed by feeding animals
dsRNA-producing bacteria as described previously
(Kamath and Ahringer, 2003)
with the following modifications. During the cloning of puf-8,
dsRNA-producing bacteria were grown on plates containing 1 mM IPTG and 5-10
adult P0 gap-1(ga133) animals were put on each plate. For the
syIs90; gap-1(ga133) strain, bacteria were induced with 6 mM IPTG,
and for all other RNAi experiments, 5-15 P0 animals were put, as L1 larvae or
as adults, on plates containing bacteria grown on 3 mM IPTG. Vulval induction
was scored in the F1 progeny at the L4 larval stage to count the number of
induced VPCs or in adults to count the percentage of Muv animals (indicated in
the table footnotes). All dsRNA-producing bacteria were from the Ahringer
library (Kamath and Ahringer,
2003), except for the fem-3 RNAi bacteria, which were a
gift from C. Eckmann.

Plasmids and PCR fusion constructs

For the puf-8::gfp translational reporter, a 3.3 kb SalI
genomic fragment containing a 1.3 kb upstream promoter fragment and the entire
C30G12.7 open reading frame was cloned into the SalI site of plasmid
pPD95.75 (a gift from A. Fire). For the fbf-2::gfp translational
reporter, a 3.73 kb BamHI genomic fragment containing a 1.5 kb
upstream promoter fragment and the entire fbf-2 open reading frame
was cloned into the BamHI site of plasmid pPD95.75. All the
dpy-7 and bar-1 promoter fusions were generated by the PCR
fusion method (Hobert, 2002).
Details on the primers used and constructions of the gfp reporters
and promoter fusions are available on request.

RESULTS

Identification of puf-8 as a negative regulator of vulval
development

Single mutants in negative regulators of vulval induction often exhibit a
wild-type vulval phenotype because these genes are mostly genetically
redundant. We therefore performed a forward genetic screen in a
gap-1(ga133) loss-of-function background to identify synthetic
mutations in additional inhibitors of vulval induction
(Canevascini et al., 2005).
gap-1 encodes a GTPase-activating protein that stimulates the
intrinsic GTPase activity of LET-60 RAS and thus inhibits the transduction of
the inductive signal (Hajnal et al.,
1997). gap-1(ga133) single mutants exhibit an elevated
activity of the EGFR/RAS/MAPK signalling pathway, yet they develop a wild-type
vulva (Fig. 1B and
Table 1, row 2). After
screening approximately 30,000 haploid genomes, we isolated 27 mutants that
displayed a synthetic multivulva (Muv) phenotype in a gap-1(ga133)
background and defined at least four complementation groups. The
ga145 mutation found in this screen caused a 60% penetrant Muv
phenotype in the gap-1(ga133) background, but no obvious vulval
phenotype as a single mutant (Table
1, rows 3 and 5). To identify additional alleles of this
complementation group, we performed a non-complementation screen (for details
see Materials and methods) that yielded a new allele (zh17)
displaying an equally penetrant synthetic Muv phenotype
(Table 1, rows 4 and 6 and
Fig. 1C). The corresponding
gene was mapped to LGII between dpy-10 and unc-4 and further
narrowed down by transformation rescue experiments to the cosmid C30G12. The
six genes on this cosmid were tested by RNAi analysis. Feeding
gap-1(ga133) animals with bacteria producing dsRNA derived from the
C30G12.7 open reading frame caused a Muv phenotype of 80% penetrance
(Table 1, row 8 and
Table 2, row 7). This gene has
previously been named puf-8, as it encodes one of the two C.
elegans Pumilio homologues (Wickens
et al., 2002). Sequencing the puf-8 coding region
revealed a stop mutation at position 485 of the ORF (CAA to TAA) before the
PUF repeats in zh17, and a G to A (GGA to AGA) transition at position
1174, replacing glycine 317 with arginine in the fourth PUF repeat in
ga145 animals (Fig.
1A). The glycine mutated in ga145 is conserved in PUF-9,
Drosophila Pumilio and the vertebrate PUF proteins. This glycine is
adjacent to an asparagine residue that is directly involved in binding to the
target mRNA (Opperman et al.,
2005). In addition to the vulval phenotype, both puf-8
alleles we isolated showed the same partially penetrant sterile phenotype at
20°C as the puf-8(ok302) deletion strain
(Fig. 1A)
(Subramaniam and Seydoux,
2003), and the puf-8(ok302) deletion caused a Muv
phenotype in a gap-1(ga133) background of similar penetrance to
zh17 or ga145 (Table
1, row 7). Thus, zh17 and ga145 are strong
reduction-of-function or null alleles of puf-8.

PUF proteins that negatively regulate vulval development. (A)
Intron-exon structure and alleles of puf-8, fbf-1 and fbf-2.
White boxes indicate the 5′UTRs, white boxes with arrowheads the
3′UTRs, grey boxes the coding regions and black boxes the PUF repeats.
(B-E) Nomarski images of the vulval cells in L4 larvae of (B)
gap-1(ga133), (C) puf-8(zh17); gap-1(ga133), and of (D,E)
fbf-1(ok91) fbf-2(q704); gap-1(ga133) animals. In all panels,
anterior is to the left and ventral is to the bottom. Note the ectopic
induction of P4.p and P8.p (arrows in C,D,E). Arrowhead in E indicates an
example of defects in the 2° cell lineage generated by P5.p resulting in
the detachment of the P5.p descendants from the cuticle in a fbf-1(ok91)
fbf-2(q704); gap-1(ga133) larva. Scale bar: 10 μm.

Genetic interaction of puf-8 with the EGFR/RAS/MAPK
pathway

We examined the genetic interaction of puf-8(zh17) with mutations
that either reduce or increase the activity of the EGFR/RAS/MAPK signalling
pathway. puf-8(zh17) partially suppressed the vulvaless (Vul)
phenotype caused by mutations in lin-2, lin-7, lin-10 and
let-60, which reduce but do not inactivate the inductive signal
(Table 1, rows 9-16)
(Kaech et al., 1998). We also
combined puf-8(zh17) with mutations in inhibitors of the
EGFR/RAS/MAPK pathway such as ark-1, sli-1 or lin-15 that
exhibit a wild-type or only a very weak Muv phenotype as single mutants
(Herman and Hedgecock, 1990;
Hopper et al., 2000;
Jongeward et al., 1995;
Yoon et al., 1995). With each
of these mutations, puf-8(zh17) caused a synthetic Muv phenotype as
described above for gap-1(ga133)
(Table 1, row 6 and rows
17-22). Thus, puf-8 either encodes a negative regulator of the
EGFR/RAS/MAPK pathway, or alternatively, puf-8 regulates the
competence of the VPCs to respond to the inductive signal.

PUF-8::GFP is expressed in vulval cells and the surrounding
epidermis

To analyze the expression pattern of PUF-8, we constructed a translational
puf-8::gfp reporter by fusing a genomic DNA fragment covering 1.3 kb
of 5′ regulatory sequences up to the next gene and the entire
puf-8 coding sequence to a GFP cassette
(Fig. 2A). PUF-8::GFP was
expressed in various tissues including the pharyngeal muscles, the hypodermis,
the ventral cord motor neurons (not shown) and the vulval cells
(Fig. 2B-J and Fig. S1A in the
supplementary material). Before vulval induction in L2 larvae, PUF-8::GFP was
expressed in all six vulval precursor cells at equal levels
(Fig. 2B,C and row with Pn.p
cells in Fig. S1A in the supplementary material). After vulval induction in
early L3 larvae, PUF-8::GFP was upregulated in the descendants of the 3°
distal VPCs (P3.p, P4.p and P8.p), while expression faded in the 1° and
2° descendants of the proximal VPCs (P5.p, P6.p and P7.p,
Fig. 2D-J, Fig. S1A in the
supplementary material, rows Pn.px to Pn.pxxx). In addition, PUF-8::GFP
expression was detected in the VulC sublineage of the 2° cells at the
Pn.pxxx stage (inset in Fig.
2H,J and Fig. S1A in the supplementary material).

We hypothesized that the increase in PUF-8::GFP expression in the
descendants of the distal 3° VPCs might occur because these cells fuse
with the hyp7 hypodermis that also expresses PUF-8::GFP. To test if the
upregulation of PUF-8::GFP in the descendants of the 3° VPCs is a
consequence of their fusion with hyp7, we examined PUF-8::GFP expression in an
eff-1(hy21) background, in which no cell fusions occur
(Mohler et al., 2002). Since
eff-1(hy21) animals exhibit excess vulval induction
(Table 1, row 23), we
additionally ablated the somatic gonad precursors Z1 and Z4 to prevent
induction by the anchor cell. In most gonad-ablated eff-1(hy21)
animals, PUF-8::GFP expression was upregulated in all VPCs and their
descendants, except for the P8.p descendants
(Fig. 2K,L and Fig. S1B in the
supplementary material). Moreover, in let-60 ras(gf) animals, in
which the distal VPCs frequently adopt the 1° or 2° induced cell
fates, PUF-8::GFP expression often remained low in the distal VPCs and their
descendants (Fig. S1C in the supplementary material)
(Beitel et al., 1990). We
conclude that PUF-8::GFP is upregulated in the descendants of VPCs that have
adopted the uninduced 3° cell fate independently of their fusion with
hyp7.

fbf-1 and fbf-2 negatively regulate vulval
development

To examine whether additional C. elegans PUF proteins besides
PUF-8 play a role in regulating vulval development, we performed an RNA
interference (RNAi) analysis by feeding rrf-3(pk1426); gap-1(ga133)
animals with dsRNA-producing bacteria derived from the other puf
genes (Kamath and Ahringer,
2003). The rrf-3(pk1426) mutation was used to increase
the sensitivity for RNAi (Simmer et al.,
2002). Of the six other PUF proteins that were tested, RNAi
against fbf-1 and fbf-2 induced a penetrant Muv phenotype,
whereas RNAi against puf-9, which is most similar to puf-8,
did not cause a Muv phenotype (Table
2, rows 1-8). Because of the high degree of sequence similarity
between the two fbf genes (over 90% identity at the nucleotide
level), RNAi against either fbf gene most likely reduces both
fbf-1 and fbf-2 expression. We therefore tested whether
fbf-1 or fbf-2 single mutants or only the fbf-1
fbf-2 double mutant show a Muv phenotype when combined with
gap-1(ga133). fbf-1(ok91); gap-1(ga133) and fbf-2(q738);
gap-1(ga133) animals both developed a wild-type vulva, but
fbf-1(ok91) fbf-2(q704); gap-1(ga133) triple mutants showed a strong
Muv phenotype (Fig. 1D,E and
Table 2 rows 9-14).
Interestingly, even in a gap-1(+) background fbf-1(ok91)
fbf-2(q704) double mutants were weakly Muv
(Table 2, row 13). Finally, we
tested for a possible redundancy among the puf genes by performing
puf-3, puf-5, puf-7, puf-8 and puf-9 RNAi in the
puf-8(zh17) and fbf-1(ok91) fbf-2(q704) backgrounds, but
observed no synthetic Muv phenotypes among the other PUF genes (data not
shown). Thus, besides puf-8 the two fbf genes encode
functionally redundant negative regulators of vulval development.

fbf-1 and fbf-2 inhibit specification of the 1°
vulval cell fate

We next determined whether PUF-8 or the FBF proteins regulate the
specification of the 1° vulval cell fate using the egl-17::yfp
reporter as a marker for the 1° cell fate
(Inoue et al., 2002).
egl-17 encodes a fibroblast growth factor (FGF) homolog that is
normally expressed in P6.p and its descendants from the time of induction
until the Pn.pxx stage (Fig.
3A,B) (Burdine et al.,
1998; Inoue et al.,
2002). In L4 larvae at the Pn.pxxx stage, EGL-17::YFP expression
disappears in the 1° cells and appears in the VulC and VulD cells of the
2° lineage (Fig. 3C,D)
(Burdine et al., 1998;
Inoue et al., 2002). Both the
early (1° fate-specific) and late (2° subfate-specific) EGL-17::YFP
expression depend on inductive signalling
(Burdine et al., 1998).

We observed a slight expansion of the early, 1°-specific EGL-17::YFP
expression in gap-1(ga133) animals causing the descendants of P5.p
and P7.p and occasionally also of P8.p to express EGL-17::YFP
(Fig. 3E,F), although,
gap-1(ga133) mutants exhibit normal vulval induction and correct
2° cell fate specification in P5.p and P7.p
(Fig. 3G,H).

Surprisingly, in puf-8(zh17); gap-1(ga133) double mutants or
puf-8 RNAi-treated gap-1(ga133) animals we observed no
increase - and sometimes even a reduction - in the 1°-specific EGL-17::YFP
expression in the proximal VPC descendants compared to gap-1(ga133)
single mutants (Fig. 3J,K).
Moreover, the descendants of P5.p and P7.p adopted a proper 2° cell fate,
as they generated seven descendants that exhibited a normal morphology and a
normal EGL-17::YFP expression pattern in the VulC and VulD subfates (compare
Fig. 3G with L). In the distal
cells (the P3.p, P4.p and P8.p descendants) we observed only a very mild
increase in the early, 1°-specific or the late, 2°-specific
EGL-17::YFP expression that did not match the frequency of ectopic vulval
induction observed in this background (Fig.
3J-M). However, it should be noted that also in other mutant
backgrounds such as let-60(n1046gf) the frequency and strength of
ectopic EGL-17::YFP expression does not mirror the level of ectopic vulval
induction (Burdine et al.,
1998).

In contrast to puf-8 mutants, fbf-1(ok91) fbf-2(q704);
gap-1(ga133) triple mutants displayed a clear upregulation of the early,
1°-specific EGL-17::YFP expression in all VPCs and their descendants
(Fig. 3N,O). Especially in the
descendants of P5.p and P7.p, the 1°-specific EGL-17::YFP expression was
much stronger than in gap-1(ga133) single mutants. In addition to the
late EGL-17::YFP expression in the ectopically induced pseudovulvae,
fbf-1(ok91) fbf-2(q704); gap-1(ga133) mutants also exhibited an
expansion of the 2°-specific EGL-17::YFP expression to 2° subfates
that normally do not express the marker (e.g. VulA and VulB in
Fig. 3P,Q). This aberrant
EGL-17::YFP expression pattern within the 2° lineage was accompanied by
morphological changes of the P5.p and P7.p descendants that are characteristic
of a partial transformation towards the 1° fate (note the detachment of
the P5.p descendants in Fig. 1E
and Fig. 3P)
(Berset et al., 2005). Such
defects in the 2° cell lineage were only rarely observed in
puf-8(zh17); gap-1(ga133) animals
(Fig. 3M).

Thus, PUF-8 and the FBF proteins perform clearly distinct roles during
vulval cell fate specification. FBF-1 and FBF-2 inhibit 1° fate-specific
gene expression and are required for proper 2° fate execution in P5.p and
P7.p, whereas PUF-8 does not regulate 1°-specific gene expression and
appears to regulate vulval induction through a different mechanism.

gld-1 is an FBF target during vulval development

Since PUF proteins function as translational repressors, the Muv phenotype
caused by puf-8 and fbf-1 and fbf-2 mutations is
probably caused by enhanced translation of their target mRNAs. Thus, RNAi
against a target mRNA that encodes a positive regulator of vulval development
should suppress the Muv phenotype of puf-8(zh17); gap-1(ga133) and/or
fbf-1(ok91) fbf-2(q704); gap-1(ga133) mutants. In the germline,
gld-1 and fem-3 are direct FBF targets that function in
mitosis/meiosis and sperm/oocyte decision, respectively
(Crittenden et al., 2002;
Zhang et al., 1997). No
targets of PUF-8 have so far been found. RNAi against gld-1
suppressed the fbf-1(ok91) fbf-2(q704); gap-1(ga133) but not the
puf-8(zh17); gap-1(ga133) Muv phenotype, whereas RNAi against
fem-3 had no effect on the Muv phenotype of either strain
(Table 2, rows 15-20). Thus,
the FBF proteins negatively regulate vulval induction by repressing, among
others, gld-1 expression. PUF-8, however, appears to act through a
distinct set of yet unknown target genes.

PUF-8::GFP and FBF-2::GFP expression during vulval development.
(A) Structure of the translational puf-8::gfp and
fbf-2::gfp reporters. (B,D,F,H)
Time-course analysis of PUF-8::GFP expression in the vulval cells from the L2
until the L4 stage with (C,E,G,J) the
corresponding Nomarski images. For a semi-quantitative analysis of the
expression patterns, see Fig. S1 in the supplementary material.
(K,L) PUF-8::GFP expression in gonad-ablated
eff-1(hy21) animals, and the corresponding Nomarski image. Note that
despite the extra round of cell divisions in P4.p and P5.p descendants of
gonad-ablated eff-1 mutants no vulval differentiation was observed.
(M-R) FBF-2::GFP expression, and the corresponding Nomarski images,
from the early L3 until the L4 stage. In all panels, anterior is to the left
and ventral is to the bottom. Scale bars: in C,L,N and in the inset of J, 10μ
m.

puf-8 controls the timing of 3° cell fusions

The upregulation of PUF-8::GFP in the distal 3° vulval cells raises the
possibility that PUF-8 might regulate the competence of the distal vulval
cells to respond to the inductive signal. Since the 3° cell fate is only
sealed after the Pn.px cells have fused with hyp7
(Wang and Sternberg, 1999),
the puf-8(lf) mutations might allow distal vulval cells to stay
unfused and hence receive the inductive signal over a longer time period,
which in combination with a second mutation in a negative regulator of the
EGFR/RAS/MAPK pathway would result in excess vulval induction.

To observe the timing of vulval cell fusions, we used the
ajm-1::gfp reporter, which labels the adherens junctions of the VPCs
and their descendants as long as they have not fused with hyp7
(Mohler et al., 1998). In
wild-type animals, the uninduced distal VPCs divide once and then rapidly fuse
with hyp7. Therefore, in the majority of wild-type larvae we analyzed at the
Pn.px stage, the descendants of P3.p, P4.p and P8.p had already fused with
hyp7 as demonstrated by the loss of AJM-1::GFP staining
(Fig. 4A-C). In
puf-8(zh17) mutants, however, the fusion of P4.p and P8.p descendants
was significantly delayed, as in approximately 50% of the animals AJM-1::GFP
staining was still present in P4.px and P8.px
(Fig. 4D-F). Note that despite
the delay in cell fusion puf-8(zh17) single mutants never showed
ectopic induction of the distal VPCs (Table
1, row 4). In fbf-1(ok91) fbf-2(q704) mutants, P4.p and
P8.p descendants were unfused in approximately 20% of the cases
(Fig. 4G-J). Since 28% of
fbf-1(ok91) fbf-2(q704) double mutants exhibit a Muv phenotype in a
gap-1(+) background (Table
2, row 13), the distal cells were probably unfused because they
had adopted a 1° or 2° vulval cell fate in these animals. PUF-8
therefore inhibits vulval development by promoting the fusion of the 3°
cells with the surrounding hyp7 hypodermis.

fbf-1 and fbf-2 inhibit 1° cell fate specification.
Analysis of EGL-17::YFP expression in mid-L3 larvae at the Pn.px or Pn.pxx
stage (left side) and in L4 larvae at the Pn.pxxx stage (right side).
(A-D) Wild-type, (E-H) gap-1(ga133), (J-M)
gap-1(ga133); puf-8 RNAi and (N-Q) fbf-1(ok91)
fbf-2(q704); gap-1(ga133) larvae. In all panels, anterior is to the left
and ventral is to the bottom. In the graphs, white indicates no EGL-17::YFP
expression, grey low expression and black high expression. The arrows in L and
P indicate ectopic induction of distal vulval cells; the arrowhead in P
indicates an example with expanded EGL-17::YFP expression in VulA and VulB,
and the resulting defect in the 2° fate execution. Scale bars: in A,C, 10μ
m.

Similar to puf-8(lf), a mutation in the effector of cell fusion
eff-1 that blocks all cell fusions causes a weak Muv phenotype that
was further enhanced by the gap-1(ga133) background
(Table 1, rows 23 and 24)
(Mohler et al., 2002).
However, it should be noted that eff-1(hy21); gap-1(ga133) double
mutants display a weaker Muv phenotype than puf-8(zh17); gap-1(ga133)
animals (Table 1, compare rows
6 and 24), indicating that puf-8 is likely to have additional
functions besides controlling the timing of 3° cell fusions.

fbf-1 and fbf-2 act in the germline and in the
soma

Thompson et al. (Thompson et al.,
2006) recently reported that feminized fbf-1 fbf-2
mutants (i.e. fbf-1 fbf-2; fog-1 or fbf-1 fbf-2; fog-3
triple mutants) display a strong Muv phenotype that is completely suppressed
by ablation of the germ cell precursors Z2 and Z3. This observation indicated
that fbf-1 and fbf-2 inhibit vulval induction in a non
cell-autonomous manner, probably by repressing the translation of a positive
regulator of vulval development in the germ cells. We performed similar gonad
precursor cell ablations, but used the fbf-1(ok91) fbf-2(q704);
gap-1(ga133) background. Ablation of Z2 and Z3 resulted in a partial
suppression of the Muv phenotype (Table
3, row 3 and Fig. S2B in the supplementary material), and ablation
of the somatic gonad precursors Z1 and Z4, which give raise to the AC,
resulted in a suppression of the Muv phenotype to nearly wild-type levels of
vulval induction (Table 3, row
4 and Fig. S2C in the supplementary material). Even after ablation of all four
gonad precursor cells (Z1 to Z4), we observed gonad-independent vulval
induction in 19% of the animals (Table
3, row 5 and Fig. S2D in the supplementary material). Since the
gap-1(ga133) mutation alone does not cause any gonad-independent
vulval induction (Hajnal et al.,
1997), fbf-1 and fbf-2 inhibit vulval
differentiation not only by repressing specific target genes in the germ cells
but also in somatic cells outside of the gonad. Supporting this hypothesis, a
translational FBF-2::GFP reporter showed an expression pattern similar to the
PUF-8::GFP pattern described above. Expression of FBF-2::GFP was first
observed at the Pn.px stage in the 3° descendants of the distal VPCs, and
it persisted throughout the L4 stage (Fig.
2A,M-R and Fig. S1D in the supplementary material).

puf-8, fbf-1 and fbf-2 act in the vulval cells

We next sought to identify the somatic tissue in which puf-8 and
fbf-1 and fbf-2 act. Since puf-8::gfp and
fbf-2::gfp are both expressed in the vulval cells as well as in the
hyp7 hypodermis, we tested whether puf-8, fbf-1 and fbf-2
act cell-autonomously in the VPCs and their descendants or non
cell-autonomously in hyp7. To distinguish between these two possibilities, we
expressed puf-8 and fbf-2 under the control of the
hypodermal dpy-7 promoter (e.g. Pdpy-7::puf-8)
(Gilleard et al., 1997), and
each of the three genes under control of a 3.1 kb bar-1 promoter
fragment that drives expression in the vulval cells, the gonadal sheath cells
and in the adult seam cells (e.g. Pbar-1::puf-8)
(Natarajan et al., 2004).
Neither the sheath cells nor the seam cells are in contact with the vulval
cells, making it very unlikely that expression of a gene in these tissues
could affect vulval induction. None of the three
Pdpy-7::puf-8 transgenes tested caused a significant
rescue of puf-8(ok302); gap-1(ga133) Muv phenotype, but two out of
three Pbar-1::puf-8 lines exhibited partial rescue, and
the third line showed a weak reduction of the Muv phenotype
(Table 4, rows 5-11). It should
be noted that even injection of a cosmid spanning the entire puf-8
locus never gave complete rescue of the Muv phenotype
(Table 4, rows 1-4). Moreover,
co-injection of Pbar-1::puf-8 with
Pdpy-7::puf-8 did not cause a stronger rescue than
injection of Pbar-1::puf-8 alone (data not shown).

puf-8 regulates the fusion of the distal vulval cells.
Vulval cell fusion was analyzed at the Pn.px stage using AJM-1::GFP as a cell
junction marker for unfused cells. (A-C) Wild-type, (D-F)
puf-8(zh17) single mutants and (G-J) fbf-1(ok91)
fbf-2(q704) double mutants. In all panels, anterior is to the left and
ventral is to the bottom. In the graphs, white represents fused Pn.px cells,
grey indicates fusing Pn.px cells that have started to dissolve their
junctions as can be seen for the P8.px cells in G, and black indicates unfused
cells with intact AJM-1::GFP-positive junctions. Note that the fraction of
unfused cells in fbf-1(ok91) fbf-2(q704) double mutants matches the
frequency of ectopically induced distal cells that give rise to the 28%
penetrant Muv phenotype (see Table
2, row 13). Scale bar in B: 10 μm.

Similarly, all but one of the Pbar-1::fbf-1 and
Pbar-1::fbf-2 transgenes reduced the penetrance of the
fbf-1(ok91) fbf-2(q704); gap-1(ga133) Muv phenotype from 90% down to
55-60%, and only one of the three Pdpy-7::fbf-2 transgenes
had a slightly significant effect (Table
4, rows 12-21). The incomplete rescue with the different
constructs is consistent with the model that fbf-1, fbf-2 as well as
puf-8 have an additional focus in the germline, since the multicopy
extrachromosomal arrays we used for these experiments are normally silenced in
the germ cells. Thus, puf-8, fbf-1 and fbf-2 negatively
regulate vulval development at least partly in the VPCs or their
descendants.

DISCUSSION

PUF proteins control somatic development

Translational repressors of the Pumilio/FBF (PUF) family regulate various
aspects of germ cell development in C. elegans by controlling the
translation of maternally provided mRNAs
(Crittenden et al., 2002;
Zhang et al., 1997). Here, we
show that three of the eleven C. elegans PUF genes also function in
the soma to control cell fate specifications during larval development. In
particular, we have found that PUF-8, FBF-1 and FBF-2 negatively regulate
vulval development in the hermaphrodite. Like most previously identified
inhibitors of vulval development, single mutants in one of these three
puf genes do not change the normal pattern of vulval cell fates.
However, when combined with another mutation in an inhibitor of the inductive
EGFR/RAS/MAPK pathway, puf-8 or fbf mutants exhibit a
hyperinduced multivulva phenotype. Genetic epistasis analysis indicates that
fbf-1 and fbf-2 perform a redundant function to inhibit
1° vulval fate specification, whereas puf-8 plays a distinct role
in regulating the temporal competence of the vulval cells to respond to the
inductive and lateral signals.

PUF-8 regulates the temporal competence of the vulval cells

Loss-of-function mutations in puf-8 partially suppress the Vul
phenotype caused by mutations that reduce but do not inactivate the
EGFR/RAS/MAPK signalling pathway. Although this observation does not prove a
direct involvement of PUF-8 in regulating the inductive EGFR/RAS/MAPK
signalling pathway, it indicates that in the absence of PUF-8 lower levels of
inductive signal are sufficient to induce vulval differentiation. A PUF-8::GFP
reporter transgene is initially expressed in all VPCs at equal levels, but
after vulval induction PUF-8::GFP expression increases in the descendants of
the distal VPCs (P3.p, P4.p and P8.p) that have adopted the 3° fate. This
expression pattern correlates well with the observed delay in the fusion of
the distal 3° cells with the hyp7 hypodermis in puf-8 mutants.
All vulval cells are competent to respond to the inductive AC and lateral
Notch signals until they fuse with hyp7
(Wang and Sternberg, 1999).
Even after the first round of vulval cell divisions, a single pulse of MAPK
activity can reprogram a 2° or 3° cell to adopt the 1° cell fate
(Berset et al., 2005). It thus
appears that by promoting the fusion of the 3° cells with hyp7, PUF-8
limits the time period during which the vulval cells can receive and integrate
the vulval patterning signals. In the absence of PUF-8, the vulval cells can
receive the inductive signal over a longer time period, which may result in
the accumulation of higher levels of activated MAPK in the distal vulval
cells. When combined with a mutation in a direct inhibitor of the
EGFR/RAS/MAPK pathway such as gap-1, this results in the ectopic
vulval differentiation and a Muv phenotype. Supporting this idea, a mutation
in the effector of cell fusion eff-1, which blocks all cell fusions,
caused a weak Muv phenotype (Mohler et
al., 2002). However, puf-8 mutants exhibit more ectopic
vulval induction in the gap-1 background than eff-1 mutants,
which points to additional functions of PUF-8 besides controlling the timing
of cell fusions.

The distal VPC descendants fuse with hyp7 shortly after they have been
born, suggesting that they exit from the cell cycle as they lose their
competence (Wang and Sternberg,
1999). The proximal vulval cells, on the other hand, go on to
divide two more times before undergoing terminal differentiation and forming a
functional vulva. It is therefore possible that PUF-8 ensures that the distal
vulval cells exit from the cell cycle immediately after they have been
generated and then fuse with hyp7. A somewhat similar function has been
proposed for the Drosophila PUF-8 orthologue Pumilio, which blocks
the cell cycle progression of the migrating pole cells during embryogenesis by
repressing cyclin B translation to prevent their premature
differentiation (Asaoka-Taguchi et al.,
1999). One could, for example, imagine that the cell cycle state
of the vulval cells and the hyp7 hypodermis needs to be coordinated to allow
the fusion between these two different cell types to occur at the right
time.

FBF-1 and FBF-2 inhibit 1° cell fate specification

In contrast to PUF-8, the FBF proteins do not regulate the timing of vulval
cell fusions, but they are more directly involved in repressing 1° vulval
fate specification. In fbf-1 fbf-2 double mutants, the expression of
the 1° fate marker EGL-17::YFP is upregulated in the ectopically induced
distal VPCs as well as in the proximal VPCs, P5.p and P7.p, which normally
adopt the 2° cell fate. puf-8 mutants, on the other hand, only
rarely exhibit ectopic expression of the 1° fate marker. This fbf-1
fbf-2 phenotype is reminiscent of the phenotype caused by mutations that
compromise the LIN-12 Notch-mediated lateral inhibition of the 1° cell
fate (Yoo et al., 2004). For
example, in ark-1 or lip-1 mutants, P5.p and P7.p frequently
express 1° cell fate marker genes. In combination with a second mutation
in an inhibitory gene, ark-1 or lip-1 mutants show similar
cell fate transformations as observed in fbf-1 fbf-2; gap-1 animals
(Berset et al., 2001;
Hopper et al., 2000). Whereas
ARK-1 and LIP-1 directly regulate EGFR and MAPK activity, respectively,
fbf-1 and fbf-2 probably inhibit vulval induction indirectly
by repressing the translation of specific target genes that activate the
EGFR/RAS/MAPK pathway.

Ablation and rescue experiments indicated that fbf-1 and
fbf-2 act in the vulval cells and in the germline in two distinct
pathways that may involve different target genes. One established target of
FBF-1 and FBF-2 in the germline is gld-1, which encodes a
translational repressor that is required for germ cells to progress through
meiosis (Crittenden et al.,
2002). Another possible FBF target proposed by Thompson et al.
(Thompson et al., 2006) is
lin-3 egf, which encodes the inductive signal that is normally
produced by the AC and repressed in the germ cells until oocyte maturation. In
feminized fbf-1 fbf-2 mutants, lin-3 egf might be
de-repressed in the meiotic germ cells, leading to excess vulval induction
from the oogenic germ cells. Inactivation of gld-1 might prevent the
overproduction of lin-3 egf because the germ cells do not enter
meiosis (Thompson et al.,
2006).

In the soma, fbf-1 and fbf-2 probably repress a different
set of target genes, since we could not observe any consistent gld-1
expression in the vulval cells, and Pn.p cell-specific RNAi against
lin-3 (Dutt et al.,
2004) did not suppress the fbf-1 fbf-2; gap-1 Muv
phenotype (data not shown). The specific targets of FBF-1 and FBF-2 in the
soma therefore remain to be identified.

PUF proteins are conserved from yeast to humans, suggesting that they
control cell fate determination in a similar way in higher organisms
(Wickens et al., 2002). It
will therefore be necessary to define the exact interplay between the PUF
family of translational regulators and the ubiquitous RTK/RAS/MAPK signalling
cascade. Translational repressors of the PUF family may turn out to play a
similar role to that of the microRNAs, in fine-tuning signalling pathways
during animal development (Giraldez et
al., 2005; Harfe et al.,
2005).

Supplementary material

Acknowledgments

We thank all lab members, A. Dutt and T. Berset for stimulating
discussions, all lab members, P. Gallant, H. Stocker and M. Gotta for comments
on the manuscript, S. K. Kim for the ga145 allele, J. Ahringer for
RNAi clones, C. Eckmann for the fem-3 RNAi clone, J. Kimble, K.
Sumbramaniam and the Caenorhabditis elegans Genetics Center for
providing some of the strains used and A. Fire for GFP reporter plasmids. This
work was supported by a grant from the Swiss National Science Foundation to
A.H. and by the Kanton Zürich.

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